The first six years of this NIH sponsored research led to the clinical introduction of Tc-99m MAG3 (mercaptoacetyltriglycine), established its dosimetry and enhanced our understanding of the use of MAG3 and OIH in renal artery stenosis. Tc-99m MAG3 has replaced OIH as the second most commonly used renal radiopharmaceutical and accounts for 19% of renal scans in the United States; however, MAG3 is quite expensive and its plasma clearance is only 50-60% that of OIH making it a suboptimal agent to estimate effective renal plasma flow. These limitations prompted us to pursue a systematic effort, guided by molecular mechanics calculations, to determine the effect of specific structural changes and charge distribution on efficient renal transport and thereby design and synthesize improved ligands. This research led to the new agent, Tc-99m N,N-1,2-ethylene-di-d-cysteine (d,d-EC) which has the highest clearance in rats of any tubular Tc-99m agent we have ever tested including MAG3; moreover, preliminary results suggest that it has a higher clearance than MAG3 in humans but it is still less than OIH. We propose to synthesize new Tc-99m, Re and Tc complexes with ligands in the successful diamido-thioether-thiolate, diamido-amino-thiolate, and diamino-dithiolate ligand classes as well as new derivatives related to MAG3. The clearance of the complexes will be measured in rats and normal volunteers. Since the high protein binding of many tubular agents, including MAG3, may limit tubular extraction and explain the suboptimal clearance of such agents compared to OIH, we propose to (a) determine the apparent protein-binding equilibrium constant for selected Tc-99m agents (b) measure their clearances in the isolated perfused rat kidney using a protein free perfusate and (c) correlate these two parameters to determine the protein binding properties which limit tubular transport. To enhance ligand design, selected Tc-99 and Re compounds will be structurally characterized by X-ray crystallography for molecular mechanics modeling. We will improve modeling calculations with inclusion of solvent and broader searches of conformational space to better characterize the structure and charge distribution associated with efficient tubular transport and optimal protein binding. The improved molecular mechanics modeling coupled with a superior understanding of protein binding has broad significance since the results can also be applied to development of non-renal radiopharmaceuticals. The higher clearance of second generation Tc-99m tubular agents will improve image quality, increase the kidney to background ratio and thereby increase the accuracy and reliability of quantitative camera based measurements. Furthermore, improved complexes will reduce the radiation dose to patients with impaired renal function, may provide a direct measurement of renal plasma flow, and will facilitate development of a new test to evaluate ureteral function. Importantly, in vivo protein binding studies should reduce the number of mammalian experiments. Finally, the availability of another Tc-99m renal tubular agent to compete with MAG3 should reduce radiopharmaceutical costs. At current levels of MAG3 use, a 30% reduction in cost would save 1.8 million dollars annually.